Medium chain fatty acid ingestion (MCFA) results in diminished fat storage in animals and humans, while similar intake of long chain fatty acids (LCFA) results in weight-gain. The goal of our project is to examine the cellular mechanisms underlying this phenomenon. Our hypothesis is that substitution of MCFA for LCFA will down-regulate the expression of adipogenic proteins, lower the lipid storage capacity of adipose cells, facilitate fatty acid release from stored fat, and probably also reduce the capacity for differentiation of adipose cell precursors. The specific aims of this study focus on: 1) the metabolic fate of MCFA and how this is influenced by LCFA, glucose and insulin, 2) the effects of MCFA on fat storage and lipolysis, and on membrane lipid composition of sub-cellular components (plasma membrane, mitochondria, and endoplasmic reticulum); 3) the effects of MCFA on the expression of adipogenic proteins during the differentiation process; and 4) changes in fat cell function as a result of long-term MCFA dietary adaptation. An integrated approach will be used to characterize the metabolic end products of MCFA, to search for unidentified end products, and to explain the excess energy expenditure that results from MCFA treatment. We will identify and quantify, the major metabolic end products of fatty acids (lipids and CO2) by NMR. 13C isotope labeled substrates will be used in appropriate experiments. This greatly enhances the selectivity and sensitivity of NMR analysis. We will compare heat generation by cells adapted to MCFA or LCFA by measuring oxygen consumption and redox state; quantify the important metabolites (acetylCoA and acetylcarnitine) by HPLC, and analyze the fatty acid compositions by GLC. If significant changes in sub-cellular membrane composition are found as a result of MCFA treatment, subsequent effects on membrane structure and fluidity will be analyzed by solid state NMR. Other standard biochemical assays will be used for the measurement of ketone bodies, total triglycerides, cholesterol, DNA, protein, etc. The mRNA products of differentiation- dependent adipogenic genes will be determined by Northern analysis. Fat cell morphology will be characterized by phase- contrast microscopy. Using this integrated approach, we anticipate developing important new information to help shed light on the molecular mechanisms of the control of obesity and obesity-related health disorders afforded through MCFA administration.